Vitamin B12 Levels of Subjects Aged 0-24 Year(s) in Konya, Turkey

ABSTRACT Research reports indicate that vitamin B12 levels show racial differences, which suggests that using the reference ranges of varied populations may lead to inaccurate results. This study aimed to determine normal serum levels of vitamin B12 among children and young people in the Konya region of Turkey. It evaluated 1,109 samples; 54 were from cord-blood and 1,055 were from healthy subjects aged 0-24 year(s), who were admitted to primary healthcare centres. The normal reference levels obtained for vitamin B12 at 2.5-97.5 percentile (P2.5-P97.5) range were 127-606 pg/mL for girls, 127-576 pg/mL for boys, and 127-590 pg/mL for the entire study group. The reported reference values for vitamin B12 in other studies were higher than the current results. Vitamin B12 levels vary from country to country; comparisons between countries may not be valid, and normal levels for each population should be obtained.


INTRODUCTION
Vitamin B 12 is a water-soluble vitamin that is an essential co-factor in some biochemical reactions and required for the synthesis of both RNA and DNA. Its deficiency may cause disorders, especially in the haematologic, neurologic, and gastrointestinal systems. Deficiency in infancy may lead to mental retardation and may have lifelong effects (1).
Reports indicate that vitamin B 12 levels show racial differences; thus, using the reference ranges of varied populations may lead to inaccurate results (2-6). Therefore, normal levels that are valid for each population should be obtained. To the authors' knowledge, no research in the literature addressed reference ranges of vitamin B 12 for the population of Konya region of Turkey. The authors designed this work to determine the normal levels of vitamin B 12 in this region.

MATERIALS AND METHODS
The study was conducted during May 2006 to

Vitamin B 12 Levels of Subjects Aged 0-24 Year(s) in Konya, Turkey
educational status, and monthly income of parents. The second section queried the medical history (disease, operations, drugs), and the third section established the nutritional status of the study group, with questions regarding fish, chicken, or red meat consumption in the previous three days and the frequency of seafood, red meat and offal consumption. Since the most important source of vitamin B 12 is red meat, consumption of red meat at least twice per week was the standard for inclusion into the study, and the subjects consuming less than that were excluded. The third section of the questionnaire asked also about the foods normally consumed at breakfast. In children who were breastfed, the study considered the nutritional status of the mother. When essential, the results of a physical examination informed the decision of including a patient. As the study was designed to determine the normal levels of vitamin B 12 , those considered to have nutritional deficiency were not included. Other exclusion criteria were: The study did not investigate the exact presence of conditions that affect vitamin B 12 levels as this requires numerous analyses that could have rendered the study impracticable in terms of time and financial cost. As in many similar studies, the declarations of subjects and parents were taken into account (7-9).
As vitamin B 12 levels are not affected by daily nutrition and do not vary throughout the day, samples were obtained between 08:00 am and 16:00 pm (10). Sera were separated by centrifugation and stored at -20 o C until the day of assay. The Biochemistry Laboratory of Selcuk University Meram Medical Faculty measured the levels of vitamin B 12 with original Beckman kits (Beckman Coulter, CA, USA) by the chemiluminescent method on UniCel DXI 800 Access immunoassay device (Beckman Coulter, CA, USA). The recommended reference interval by the manufacturer was 180-914 pg/mL. The intra-assay precision values of the kit were 5% and 11.4% for 88 pg/mL and 914 pg/mL respectively. The inter-assay precision values were 8.5% and 11.4% respectively. The analytic sensitivity of the kits was 50 pg/mL and analytic specificity was 99.5%.

Statistical analysis
Statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS) (version 13.0). Data were summarized as mean±standard deviation and median. While Mann-Whitney Utest was used in comparing two groups, one-way variance analysis was used when more than two groups were compared. Association of parameters was determined by Spearman's correlation coefficient. A value of p<0.05 was considered to be statistically significant.
An evaluation of major sources for intake of vitamin B 12 showed that intake rates of fish and offal were low. Cheese, yogurt and egg intakes were high at breakfast. Consumption frequency of red meat was at least two times per week for the whole study group (Table 1).

Vitamin B 12 levels
Mean vitamin B 12 levels in boys, girls, and the general population (aged 1 month to 24 years) were 262.7±130 pg/mL, 263±124 pg/mL, and 263±127 pg/mL respectively. Table 2 gives the vitamin B 12 levels by age-group; Table 3 shows the results for cord-blood and newborns. Evaluation of these results revealed that vitamin B 12 levels of both male and female subjects in the 1-5 year(s) old and the 6-11 years old groups were significantly higher than levels among the newborns (p<0.05 vs p<0.01). The levels in 1-5 year(s) old males and both 1-5 year(s) old and 6-11 years old females were significantly higher than the levels found in the 12-17 years old subjects (p<0.05). Additionally, the levels in the 1-5 year(s) old and the 6-12 years old females were significantly higher than the levels in 18-24 years old subjects (p<0.01).
Evaluation of the subjects according to gender revealed that vitamin B 12 levels were significantly higher in females at the age of 7 and 14 years and in males at the age of 21 years when compared with the opposite gender (p<0.05). Considering correlations of vitamin B 12 levels with age, height, weight, and BMI, a negative association was obtained with each (p<0.001).
Associations of educational status with vitamin B 12 levels in fathers revealed that children of fathers who had high school education had significantly higher levels than children of fathers who were non-literate or who had a primary school education (286.6±147.5 pg/mL vs 248.2±133.7 pg/mL and 255.8±140.5 pg/mL respectively, p<0.05) while children of mothers with a university degree had significantly higher vitamin B 12 levels than children of non-literate mothers (316.4±160.0 pg/mL vs 275.1±197.6 pg/mL respectively, p<0.05). Vitamin B 12 levels showed no significant differences for the parents' occupations or monthly incomes.

Dietary habits
Assessment of data showed no statistically significant difference for the frequency of red meat consumption. A significant difference did exist between subjects who never consumed fish and those who consumed it once or more per month (238.7±111.9 pg/mL vs 270.4±144.7 pg/mL and 285.0±151.4 pg/mL respectively, p<0.05). Vitamin B 12 levels showed no difference between those who did or did not consume meals, including meat in the last three days. However, comparison of levels in subjects who received meals, including meat in the last three days, showed that subjects consuming fish had higher levels than did subjects receiving chicken (290.9±146.9 pg/mL vs 242.4±115.0 pg/ mL respectively, p<0.05). No significant differences existed among the groups in terms of the frequency of offal consumption.
Subjects consuming butter at breakfast had higher vitamin B 12 levels than those who never ate breakfast (303.9±187.9 pg/mL vs 220.0±89.1 pg/mL respectively, p<0.05). Subjects consuming milk at breakfast had significantly higher vitamin B 12 levels when compared with those drinking tea or nothing (297.5±145.1 pg/mL vs 252.6±136.8 pg/mL and 244.8±75.3 pg/mL respectively, p<0.05).

DISCUSSION
The reference levels of vitamin B 12 found in the current study were different from the levels found in studies conducted in other countries (  (13).
The dissemination of health services and increase in general knowledge among the population has served to increase the number of those benefiting from preventive health services. This has facilitated a decrease in the incidence of various diseases, such as avitaminosis A and D, which were previously prevalent. At the same time, vitamin B 12 deficiency has attracted increased attention. Vitamin B 12 deficiency was found in 11% of the Guatemalan school children (15). Vitamin B 12 deficiency is also not rare in Turkey (16).
A review of studies conducted on vitamin B 12 levels among children show different results as shown in Table 4. In the study of Davis et al., infants of 4-37 weeks had higher 95% CI limit and mean value ; According to the international unit system, conversion coefficient pg/mL to pmol/L is 0.74' (pmol/L=pg/mLx0.74) and lower 5% limit and mean value of vitamin B 12 when compared with our results (17). The values obtained in other studies (mean and percentage limit values), however, were higher than the values found in this study (8,13,(18)(19)(20)(21)(22)(23)(24). These differences might be attributed to several factors, particularly the criteria and requirements for inclusion into the study. Davis et al. accepted only good health status and being breastfed as requirements for inclusion into the study (17). The vitamin B 12 levels in the infants' blood and their umbilical cord are closely related to the level in the mothers' blood (16,25). However, the drugs containing vitamin B 12 used during pregnancy and after giving birth and similar drugs that can be given to the infants have an increasing effect on vitamin B 12 levels. Among other researchers, only Huemer et al. indicated the absence of any vitamin drug as a requirement for inclusion in their study (22). Thus, differences in the inclusion criteria for study subjects could affect the study results.
Another reason for the differences observed between other studies and the current study can be nutritional habits. Disorders relating to excessive nutrition, such as obesity, are quite common in western societies; people in those societies are wellnourished, even excessively so. In Finland, subjects have been advised to take two mcg of vitamin B 12 daily, yet their daily vitamin B 12 intake was 7.4-11 mcg (12). The current study accepted consumption of red meat twice per week as an adequate nutritional sign of vitamin B 12 intake. The findings regarding the nutritional habits of the participants indirectly confirmed that they had consumed red meat at least twice per week. If participants reported that they had consumed red meat at least twice per week despite they had actually consumed less, their vitamin B 12 levels would be low. In that case, the vitamin B 12 levels in participants who stated that they had consumed meat daily and preferred fish and food of animal origin at breakfast should have shown a significant difference when compared with those who did not prefer these types of food. However, examination of the results showed no significant difference. Meat consumed in this quantity, together with the consumption of other foods of animal origin, maintained an adequate vitamin B 12 level but did not lead to a significant difference. This also shows that subjects who stated they consumed red meat at least twice per week were reporting accurately.
The following two points can help explain the low values observed in our study: (i) Ethnic diversity: Some researchers have noted differences in vitamin B 12 levels between the members of the white and black races (4,11,12). Kwee et al. reported vitamin B 12 levels of 382±131.3 pg/mL and 546±197.5 pg/mL in healthy white and black females respectively. They stated that the difference between the two races was significant (4). A similar characteristic might affect other societies, and one might question whether ethnic characteristics have affected the results of the current study. It would be presumptuous to provide a definitive answer to this question in view of the inadequate number of studies on the issue. However, evidence exists that would lead us to think the opposite. In a study conducted in Australia, 56 of the participants were Turkish (22). In that study, while the vitamin B 12 level of Turkish children was reported to be 592±70 pg/mL, that of Australian children was 469±79 pg/ mL. This finding shows that Turkish people living in the same region with Australians and with similar opportunities for nutrition do not have low vitamin B 12 levels.
(ii) Vitamin B 12 content of nutriments: The source of vitamin B 12 is food of animal origin. Animals do not produce this vitamin themselves. Animals eat food containing B 12 -producing bacteria; thus, the animals become sources of vitamin B 12 (26). One explanation of low levels of vitamin B 12 in humans could be low levels of cobalt in the soil, resulting in fewer microorganisms producing vitamin B 12 by using cobalt in the region's soil. There is insufficient information to confirm or reject such a hypothesis.
Another reason for the different results among studies might be the differences between the methodologies used. The current study utilized the Beckman kits that are used in the biochemistry laboratory of the hospital for measuring vitamin levels. The reference range of 127-590 pg/mL obtained in the study was different from the reference range of the kits (180-914 pg/mL). The study by Christenson et al., conducted on 154 healthy subjects, investigated the reference ranges using two different kits. Reference ranges were found to be 116-817 ng/L with the SimulTRAC-S kit and 205-810 ng/L with the Quantaphase kit (27). It is remarkable that there is a substantial difference between these results. The reference ranges of the kits were 180-960 and >200 ng/L respectively. Furthermore, false positivity was detected in 9% of the subjects with the first kit. Thus, Christenson et al. suggested that studies on normal range be completed in a society before assays are used in the evaluation of patients; the current authors support this idea (27). Kumar et al. compared three methods in a study conducted on the Indians, and they recommended radioisotope dilution assay as an accurate procedure for determining vitamin B 12 levels (28).
In the current study, we found that when the factors that can affect vitamin B 12 levels such as age, weight, height, and BMI increased, vitamin B 12 levels were significantly lowered. In line with the current findings, others have reported that vitamin B 12 levels significantly decrease with age (6,7,18,22,24,29). When subjects were stratified according to gender, it was found that vitamin B 12 levels were significantly higher in females at the age of 7 and 14 years and in males at the age of 21 years. However, the findings about the association of gender and vitamin B 12 levels are incompatible with the literature (6,8,11,18,20,22,29).
In this study, the serum level of vitamin B 12 was not affected by the parents' occupations or the levels of family income. This finding gave rise to the thought that people living in the region had good awareness of nutritional issues. The finding that vitamin B 12 levels in children whose mothers had a university degree and whose fathers had high school education were significantly higher suggests education can be an effective factor in nutrition.
An interesting result of this research was that vitamin B 12 levels were significantly higher in subjects who consumed fish when compared with those who consumed chicken. No comparable result could be found in the literature. This situation can be explained because the vitamin B 12 level in fish is 10 times higher than in chicken (30). It is not surprising that infants taking complementary food had higher vitamin B 12 levels compared to infants who were only breastfed. Infants are generally given food prepared with milk as a supplement. The vitamin B 12 content in cow's milk is 5 to 10 times higher than that in human milk (30,31). The findings of the current study were supported by those of Davis et al. and Karademir et al. who reported that vitamin B 12 levels in infants who were only breastfed were lower when compared with levels in infants who were fed with cow's milk and formula milk (17,32).

Conclusions
The results of this research suggest that vitamin B 12 levels vary among countries and that using reference ranges of varied populations may lead to inac-curate results. Therefore, the researchers advise that it would be beneficial to achieve normal levels that are valid for each population.